Somatostatin antagonists

ABSTRACT

The present invention is directed to a somatostatin antagonist according to formula (I), wherein A 1  is an optionally substituted aromatic ∝-amino acid; A 2  is an optionally substituted aromatic ∝-amino acid; A 3  is Dab, Dap, Lys or Orn; A 4  is β-Hydroxyvaline, Ser, Hser, or Thr; A 5  is an optionally substituted D- or L-aromatic -amino acid; and Y 1  is OH, NH 2  or NHR 1 , where R 1  is (C 1-6 )alkyl; wherein each said optionally substituted aromatic -amino acid is optionally substituted with one or more substituents each independently selected from the group consisting of halogen, NO 2 , OH, CN, (C 1-6 )alkyl, (c 2-6 )alkenyl, (c 2-6 )alkynyl, (C 1-6 )alkoxy, Bzl, O-Bzl, and NR 9 R 10 , where R 9  ad R 10  each is independently H, O, or (C 1-6 )alkyl; and wherein the amine nitrogen of each of amide peptide bond and the amino group of A 1  of formula (I) is optionally substituted with a methyl group, provided that there is at least one said methyl group; or a pharmaceutically acceptable salt thereof, and to uses thereof.

BACKGROUND OF THE INVENTION

[0001] Somatostatin (SRIF), a tetradecapeptide discovered by Brazeau etal., has been shown to have potent inhibitory effects on varioussecretory processes in tissues such as pituitary, pancreas andgastrointestinal tract. SRIF also acts as a neuromodulator in thecentral nervous system. These biological effects of SRIF, all inhibitoryin nature, are elicited through a series of G protein coupled receptors,of which five different subtypes have been characterized (sstr₁-sstr₅).These five subtypes have similar affinities for the endogenous SRIFligands but have differing distribution in various tissues. Somatostatinbinds to the five distinct receptor (SSTR) subtypes with relatively highand equal affinity for each subtype. Binding to the different types ofsomatostatin subtypes have been associated with the treatment of variousconditions and/or diseases. (“sstr₂”) (Raynor, et al., MolecularPharmacol. 43:838 (1993); Lloyd, et al., Am. J. Physiol. 268:G102(1995)) while the inhibition of insulin has been attributed to thesomatostatin type-5 receptor (“sstr₅”) (Coy, et al. 197:366-371 (1993)).Activation of types 2 and 5 have been associated with growth hormonesuppression and more particularly GH secreting adenomas (Acromegaly) andTSH secreting adenomas. Activation of type 2 but not type 5 has beenassociated with treating prolactin secreting adenomas. Other indicationsassociated with activation of the somatostatin receptor subtypes areinhibition of insulin and/or glucagon for treating diabetes mellitus,angiopathy, proliferative retinopathy, dawn phenomenon and nephropathy;inhibition of gastric acid secretion and more particularly pepticulcers, enterocutaneous and pancreaticocutaneous fistula, irritablebowel syndrome, Dumping syndrome, watery diarrhea syndrome, AIDS relateddiarrhea, chemotherapy-induced diarrhea, acute or chronic pancreatitisand gastrointestinal hormone secreting tumors; treatment of cancer suchas hepatoma; inhibition of angiogenesis, treatment of inflammatorydisorders such as arthritis; retinopathy; chronic allograft rejection;angioplasty; preventing graft vessel and gastrointestinal bleeding. Itis preferred to have an analog which is selective for the specificsomatostatin receptor subtype or subtypes responsible for the desiredbiological response, thus, reducing interaction with other receptorsubtypes which could lead to undesirable side effects.

[0002] The development of potent, smaller SRIF agonists led to thediscovery of differing affinities of the various truncated ligands forthe different subtypes. It appears that Trp⁸-Lys⁹ sequence often ispresent in ligands that are recognized by the receptor. The Trp⁸-Lys⁹sequence forms part of a β-bend which is usually stabilized viasubstitution of D- for L-Trp, cyclization of the backbone, a disulfidebridge, or all constraints. One unintended consequence of suchstructural simplification, carried out before the discovery of multiplereceptor subtypes, was the loss of broad spectrum binding affinity. Thisis typified by the high type 2 but low type 1, 3, 4, and 5 affinities ofpeptides in the OCTREOTIDE® series. Thus, the many basic biologicalstudies with this type of analog failed to detect effects mediated byall but one of the somatostatin receptor types. Since then, much workhas gone into the re-introduction of broader spectrum binding intosmall, biologically stable peptides on the one hand and the developmentof peptides and peptidomimetics with discrete specificity for aparticular receptor.

[0003] We have discovered that peptide backbone constraint can beintroduced by N-alkylation of individual amino acids. This modificationlargely restricts the affected residue and the amino acid preceding itto an extended conformation. Thus, additionally blocks potentialintramolecular hydrogen bonding sites and also proteolytic enzymecleavage sites thus potentially enhancing the pharmacokinetic propertiesof a peptide. Only a few N-methyl amino acids are commercially availableand their synthesis is tedious. However, in another aspect of thepresent invention, we have discovered a procedure to N-methylatetruncated somatostatin analogs at every amino acid residue using thesolid-phase procedure, adopted from the recent publication reported byMiller and Scanlan.

SUMMARY OF THE INVENTION

[0004] In one aspect, the present invention is directed to a peptide ofthe formula (I),

A¹-cyclo{D-Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹,  (I)

[0005] wherein:

[0006] A¹ is an optionally substituted aromatic α-amino acid;

[0007] A² is an optionally substituted aromatic α-amino acid;

[0008] A³ is Dab, Dap, Lys or Orn;

[0009] A⁴ is β-Hydroxyvaline, Ser, Hser, or Thr;

[0010] A⁵ is an optionally substituted D- or L-aromatic α-amino acid;and

[0011] Y¹ is OH, NH₂ or NHR¹, where R¹ is (C₁₋₆)alkyl;

[0012] wherein each said optionally substituted aromatic α-amino acid isoptionally substituted with one or more substituents each independentlyselected from the group consisting of halogen, NO₂, OH, CN, (C₁₋₆)alkyl,(C₂₋₆)alkenyl, (C₂₋₆)alkynyl, (C₁₋₆)alkoxy, Bzl, O-Bzl, and NR⁹R¹⁰,where R⁹ and R¹⁰ each is independently H, O, or (C₁₋₆) alkyl; and

[0013] wherein the amine nitrogen of each of amide peptide bond and theamino group of A¹ of formula (I) is optionally substituted with a methylgroup, provided that there is at least one said methyl group;

[0014] or a pharmaceutically acceptable salt thereof.

[0015] In one embodiment the invention features peptides of formula (I)wherein:

[0016] A¹ is Cpa, 1-Nal, 2-Nal, 2-Pal, 3-Pal, 4-Pal, Phe, Tfm, Tyr orTyr(I);

[0017] A² is 2-Pal, 3-Pal, 4-Pal, Phe, Tyr or Tyr(I); and

[0018] A⁵ is Dip, 1-Nal, 2-Nal, 2-Pal, 3-Pal, 4-Pal, Phe or D-Trp;

[0019] or a pharmaceutically acceptable salt thereof.

[0020] In another embodiment the invention features a peptide of theimmediately foregoing group of peptides wherein A¹ is Cpa.

[0021] In a further embodiment the invention features a peptide of theimmediately foregoing group of peptides wherein A³ is NMeLys.

[0022] In a still further embodiment the invention features a peptide offormula (I) wherein said peptide is:

[0023] NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;

[0024] Cpa-cyclo(NMeDCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NHMe;

[0025] Cpa-cyclo(DCys-NMe3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;

[0026] Cpa-cyclo(DCys-3-Pal-NMeDTrp-Lys-Thr-Cys)-2-Nal-NH₂;

[0027] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0028] Cpa-cyclo(DCys-3-Pal-DTrp-Lys-NMeThr-Cys)-2-Nal-NH₂;

[0029] Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-NMeCys)-2-Nal-NH₂;

[0030] Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-Nme2-Nal-NH₂;

[0031] Cpa-cyclo(NMeDCys-3-Pal-DTrp-Lys-Thr-Cys)-Dip-NHMe;

[0032] Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0033] Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0034] Tfm-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0035] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;

[0036] Nal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or

[0037] 3-Pal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;

[0038] or a pharmaceutically acceptable salt thereof.

[0039] In yet a further embodiment the invention features a peptide offormula (I) wherein said peptide is:

[0040] NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;

[0041] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0042] Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0043] Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0044] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;

[0045] Nal-cyclo(DCys-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or

[0046] 3-Pal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;

[0047] or a pharmaceutically acceptable salt thereof.

[0048] In still yet a further embodiment the invention features apeptide of formula (I) wherein said peptide is:

[0049] NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;

[0050] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0051] Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0052] Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂; or

[0053] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;

[0054] or a pharmaceutically acceptable salt thereof.

[0055] In still yet a further embodiment the invention features apeptide of the immediately foregoing group of peptides wherein saidpeptide is:

[0056] Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂; or

[0057] Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;

[0058] or a pharmaceutically acceptable salt thereof.

[0059] In another aspect of the present invention is featured a methodof binding one or more somatostatin subtype receptors −1, −2, −3, −4 and−5, which comprises the step of contacting a compound of claim 1 or apharmaceutically acceptable salt thereof with one or more of saidsomatostatin subtype receptors.

[0060] In one embodiment of the immediately foregoing aspect the presentinvention features a method of binding one or more somatostatin subtypereceptors −1, −2, −3, −4 and −5 in a human subject or other animalsubject, which comprises the step of administering an effective amountof a compound of claim 1 or a pharmaceutically acceptable salt thereofto a subject in need thereof.

[0061] In another embodiment aspect of the present invention is featureda method of eliciting a somatostatin antagonist effect from a cell,wherein said cell comprises one or more somatostatin receptors, saidmethod comprising contacting said cell with an effective amount of acompound of claim 1 or a pharmaceutically acceptable salt thereof.

[0062] In another embodiment, the present invention provides a methodfor eliciting a somatostatin antagonist effect in a human subject orother animal subject, which comprises the step of administering aneffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof to a subject in need thereof.

[0063] In a further embodiment of the present invention is featured amethod of promoting the release of growth hormone in a human or animalsubject, which comprises administering to said subject an effectiveamount of a compound according to claim 1 or a pharmaceuticallyacceptable salt thereof.

[0064] In another embodiment of the present invention is featured amethod of promoting the release of insulin in a human or animal subjectin need thereof, which comprises administering to said subject aneffective amount of a compound according to claim 1 or apharmaceutically acceptable salt thereof.

[0065] In another embodiment of the present invention is featured amethod of enhancing wound healing in a human or animal subject in needthereof, which comprises administering to said subject an effectiveamount of a compound according to claim 1 or a pharmaceuticallyacceptable salt thereof.

[0066] In yet another embodiment of the present invention is featured amethod of promoting angiogenesis in a human or animal subject in needthereof, which comprises administering to said subject an effectiveamount of a compound according to claim 1 or a pharmaceuticallyacceptable salt thereof.

[0067] In another embodiment of the present invention is featured amethod of treating a disease or condition in a human or other animalsubject in need thereof, which comprises the step of administering aneffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof to said subject, wherein said disease orcondition is selected from the group consisting of short stature,cachexia, wasting, type 2 diabetes, poor circulation, and the like.

[0068] In another aspect of the present invention is featured a methodof imaging cells having somatostatin receptors which comprisescontacting said cells with an effective amount of a compound accordingto claim 1, or a pharmaceutically acceptable salt thereof, whichcomprises Tyr(I).

BRIEF DESCRIPTION OF THE DRAWING

[0069]FIG. 1 is a graph showing the in vitro inhibition of hsstr-5mediated intracellular Ca2+ mobilization.

DETAILED DESCRIPTION

[0070] One skilled in the art can, based on the description herein,utilize the present invention to its fullest extent. The followingspecific embodiments are, therefore, to be construed as merelyillustrations of the invention and are not meant to be construed aslimiting the full scope of the invention.

[0071] As is well known to those skilled in the art, the known andpotential uses of somatostatin are varied and multitudinous.Somatostatin and analogs thereof are known to be useful in the treatmentof the diseases and/or conditions listed hereinbelow. The varied uses ofsomatostatin may be summarized as follows: Cushings Syndrome (see Clark,R. V. et al, Clin. Res. 38, p. 943A, 1990); gonadotropinoma (see AmbrosiB., et al., Acta Endocr. (Copenh.) 122, 569-576, 1990);hyperparathyroidism (see Miller, D., et al., Canad. Med. Ass. J., Vol.145, pp. 227-228, 1991); Paget's disease (see, Palmieri, G. M. A., etal., J. of Bone and Mineral Research, 7, (Suppl. 1), p. S240 (Abs. 591),1992); VIPoma (see Koberstein, B., et al., Z. Gastroenterology, 28,295-301, 1990 and Christensen, C., Acta Chir. Scand. 155, 541-543,1989); nesidioblastosis and hyperinsulinism (see Laron, Z., Israel J.Med. Sci., 26, No. 1, 1-2, 1990, Wilson, D. C., Irish J. Med. Sci., 158,No. 1, 31-32, 1989 and Micic, D., et al., Digestion, 16, Suppl. 1.70.Abs. 193, 1990); gastrinoma (see Bauer, F. E., et al., Europ. J.Pharmacol., 183, 55 1990); Zollinger-Ellison Syndrome (see Mozell, E.,et al., Surg. Gynec. Obstet., 170, 476-484, 1990); hypersecretorydiarrhea related to AIDS and other conditions (due to AIDS, see Cello,J. P., et al., Gastroenterology, 98, No. 5, Part 2, Suppl., A163 1990;due to elevated gastrin-releasing peptide, see Alhindawi, R., et al.,Can. J. Surg., 33, 139-142, 1990; secondary to intestinal graft vs. hostdisease, see Bianco J. A., et al., Transplantation, 49, 1194-1195, 1990;diarrhea associated with chemotherapy, see Petrelli, N., et al., Proc.Amer. Soc. Clin. Oncol., Vol. 10, P 138, Abstr. No. 417 1991); irritablebowel syndrome (see O'Donnell, L. J. D., et al., Aliment. Pharmacol.Therap., Vol. 4., 177-181, 1990); pancreatitis (see Tulassay, Z., etal., Gastroenterology, 98, No. 5, Part 2, Suppl., A238, 1990); Crohn'sDisease (see Fedorak, R. N., et al., Can. J. Gastroenterology, 3, No. 2,53-57, 1989); systemic sclerosis (see Soudab, H., et al.,Gastroenterology, 98, No. 5, Part 2, Suppl., A129, 1990); thyroid cancer(see Modigliani, E., et al., Ann., Endocr. (Paris), 50, 483-488, 1989);psoriasis (see Camisa, C., et al., Cleveland Clinic J. Med., 57, No. 1,71-76, 1990); hypotension (see Hoeldtke, R. D., et al., Arch. Phys. Med.Rehabil., 69, 895-898, 1988 and Kooner, J. S., et al., Brit. J. Clin.Pharmacol., 28, 735P-736P, 1989); panic attacks (see Abelson, J. L., etal., Clin. Psychopharmacol., 10, 128-132, 1990); sclerodoma (see Soudah,H., et al., Clin. Res., Vol. 39, p. 303A, 1991); small bowel obstruction(see Nott, D. M., et al., Brit. J. Surg., Vol. 77, p. A691, 1990);gastroesophageal reflux (see Branch, M. S., et al., Gastroenterology,Vol. 100, No. 5, Part 2 Suppl., p. A425, 1991); duodenogastric reflux(see Hasler, W., et al., Gastroenterology, Vol. 100, No. 0.5, Part 2,Suppl., p. A448, 1991); Graves' Disease (see Chang, T. C., et al., Brit.Med. J., 304, p. 158, 1992); polycystic ovary disease (see Prelevic, G.M., et al., Metabolism Clinical and Experimental, 41, Suppl. 2, pp76-79, 1992); upper gastrointestinal bleeding (see Jenkins, S. A., etal., Gut., 33, pp. 404-407, 1992 and Arrigoni, A., et al., AmericanJournal of Gastroenterology, 87, p. 1311, (abs. 275), 1992); pancreaticpseudocysts and ascites (see Hartley, J. E., et al., J. Roy. Soc. Med.,85, pp. 107-108, 1992); leukemia (see Santini, et al., 78, (Suppl. 1),p. 429A (Abs. 1708), 1991); meningioma (see Koper, J. W., et al., J.Clin. Endocr. Metab., 74, pp. 543-547, 1992); and cancer cachexia (seeBartlett, D. L., et al., Surg. Forum., 42, pp. 14-16, 1991). Thecontents of the foregoing references are incorporated herein byreference.

[0072] The peptides of the invention are useful as antagonists to theactivity or activities of somatostatin. For example, the peptides of theinvention can be used to promote the release of growth hormone orinsulin in a subject (e.g., a mammal such as a human patient). Thus, thepeptides are useful in the treatment of physiological conditions inwhich the promotion of the release of growth hormone or insulin is ofbenefit. The peptides of the invention can also be used in enhancingwound healing or promoting angiogenesis. Further, peptides of theinvention having a Tyr(I) residue can be used to image cells containingsomatostatin receptors. Such peptides of the invention can be usedeither in vivo to detect cells having somatostatin receptors (e.g.,cancer cells) or in vitro as a radioligand in a somatostatin receptorbinding assay. The peptide of the invention can also be used as vectorsto target cells with radioactive isotopes.

[0073] Also contemplated within the scope of this invention is a peptidecovered by the above generic formula for both use in treating diseasesor disorders associated with the need to promote the release of growthhormone or insulin, and use in detecting somatostatin receptors, e.g.,radioimaging.

[0074] A compound of formula (I) or a pharmaceutically-acceptable saltthereof can be administered by oral, parenteral (e.g., intramuscular,intraperitoneal, intravenous- or subcutaneous injection, or implant),nasal, vaginal, rectal, sublingual or topical routes of administrationand can be formulated with pharmaceutically acceptable carriers toprovide dosage forms appropriate for each route of administration.

[0075] Solid dosage forms for oral administration include capsules,tablets, pills, powders and granules. In such solid dosage forms, theactive compound is admixed with at least one inert pharmaceuticallyacceptable carrier such as sucrose, lactose, or starch. Such dosageforms can also comprise, as is normal practice, additional substancesother than such inert diluents, e.g., lubricating agents such asmagnesium stearate. In the case of capsules, tablets and pills, thedosage forms may also comprise buffering agents. Tablets and pills canadditionally be prepared with enteric coatings.

[0076] Liquid dosage forms for oral administration includepharmaceutically acceptable emulsions, solutions, suspensions, syrups,the elixirs containing inert diluents commonly used in the art, such aswater. Besides such inert diluents, compositions can also includeadjuvants, such as wetting agents, emulsifying and suspending agents,and sweetening, flavoring and perfuming agents.

[0077] Preparations according to this invention for parenteraladministration include sterile aqueous or non-aqueous solutions,suspensions, or emulsions. Examples of non-aqueous solvents or vehiclesare propylene glycol, polyethylene glycol, vegetable oils, such as oliveoil and corn oil, gelatin, and injectable organic esters such as ethyloleate. Such dosage forms may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. They may be sterilized by,for example, filtration through a bacteria-retaining filter, byincorporating sterilizing agents into the compositions, by irradiatingthe compositions, or by heating the compositions. They can also bemanufactured in the form of sterile solid compositions which can bedissolved in sterile water, or some other sterile injectable mediumimmediately before use.

[0078] Compositions for rectal or vaginal administration are preferablysuppositories which may contain, in addition to the active substance,excipients such as coca butter or a suppository wax.

[0079] Compositions for nasal or sublingual administration are alsoprepared with standard excipients well known in the art.

[0080] The dosage of active ingredient in the compositions of thisinvention may be varied; however, it is necessary that the amount of theactive ingredient be such that a suitable dosage form is obtained. Theselected dosage depends upon the desired therapeutic effect, on theroute of administration, and on the duration of the treatment.Generally, dosage levels of between 25 μg/kg/day to 100 mg/kg/day ofbody weight daily are administered as a single dose or divided intomultiple doses to humans and other animals, e.g., mammals, to obtain thedesired therapeutic effect.

[0081] A preferred general dosage range is 250 μg/kg/day to 5.0mg/kg/day of body weight daily which can be administered as a singledose or divided into multiple doses.

[0082] Further, a compound of formula (I) can be administered in asustained release composition such as those described in the followingpatents. Among those formulations, 14-day or 28-day slow releaseformulations will be preferred. U.S. Pat. No. 5,672,659 teachessustained release compositions comprising a peptide and a polyester.U.S. Pat. No. 5,595,760 teaches sustained release compositionscomprising a peptide in a gelable form. U.S. Pat. No. 5,821,221 teachespolymeric sustained release compositions comprising a peptide andchitosan. U.S. Pat. No. 5,916,883 teaches sustained release compositionscomprising a peptide and cyclodextrin. International Patent ApplicationNo. PCT/US99/01180, (publication no. WO 99/38536, Aug. 5, 1999), teachesabsorbable sustained release compositions of a peptide. The contents ofthe foregoing patents and applications are incorporated herein byreference.

[0083] The use of immediate or of sustained release compositions dependson the type of indications targeted. If the indication consists of anacute or over-acute disorder, a treatment with an immediate form will bepreferred over the same with a prolonged release composition. On thecontrary, for preventive or long-term treatments, a prolonged releasecomposition will generally be preferred.

[0084] Abbreviations

[0085] The nomenclature for the somatostatin receptor subtypes is inaccordance with the recomendations of IUPHAR, in which sstr₄ refers tothe receptor originally cloned by Bruno et al., and sstr₅ refers to thereceptor cloned by O'Carroll et al.

[0086] Abbreviations of the common amino acids are in accordance withthe recommendations of IUPAC-IUB. Further, as used herein thedefinitions for certain abbreviations are as follows:

[0087] Abu=α-aminobutyric acid;

[0088] Aib=α-aminoisobutyric acid;

[0089] β-Ala=β-alanine;

[0090] Amp=4-amino-phenylalanine;

[0091] Ava=5-aminovaleric acid;

[0092] Cha=cyclohexylalanine;

[0093] Cpa=3-(4-chlorophenyl)alanine;

[0094] Dab=2,4-diaminobutyric acid;

[0095] Dap=2,3-diaminopropionic acid;

[0096] Dip=3,3′-diphenylalanine;

[0097] Gaba=γ-aminobutyric acid;

[0098] HSer=homoserine;

[0099] 1-Nal=3-(1-naphthyl)alanine;

[0100] 2-Nal=3-(2-naphthyl)alanine;

[0101] Nle=norleucine;

[0102] Nva=norvaline;

[0103] 2-Pal=3-(2-pyridyl)alanine;

[0104] 3-Pal=3-(3-pyridyl)alanine;

[0105] 4-Pal=3-(4-pyridyl)alanine;

[0106] Tfm=Trifluoromethyl; and

[0107] TfmA=4-trifluoromethylphenyl-alanine.

[0108] Tyr(I)=An iodinated tyrosine residue (e.g., 3-1-Tyr, 5-I-Tyr,3,5-I -Tyr) wherein the iodine may be a radioactive isotope, e.g., I₁₂₅,I₁₂₇, or I₁₃₁.

[0109] The following abbreviations of certain reagents also appearherein:

[0110] DBU=1,8-diazabicyclo[5.4.0]undec-7-ene;

[0111] DCM=dichloromethane;

[0112] DIC=diisopropylcarbodiimide;

[0113] DIEA=diisopropyethylamine;

[0114] DMF=dimethylformamide;

[0115] MTBD=1,3,4,6,7,8-Hexahydro-1-methyl-2H-pyrimido[1,2-a]pyrimidine;

[0116] o-NBS=2-nitrobenzenesulfonyl;

[0117] TBTU=O-Benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumtetrafluoroborate;

[0118] and

[0119] TFA=trifluoroacetic acid.

[0120] With the exception of the N-terminal amino acid, allabbreviations (e.g., Cpa for A¹) of amino acids in this disclosure standfor the structure of —NH—CH(R)—CO—, wherein R is the side chain of anamino acid (e.g., CH₃ for Ala). For the N-terminal amino acid, theabbreviation stands for the structure of (R¹R²)—N—CH(R)—CO—, wherein Ris a side chain of an amino acid and each of R¹ and R² is independentlyH or as otherwise defined herein.

[0121] An aliphatic amino acid is an α-amino acid having one or two sidechains which, independently, are hydrocarbons, e.g., a straight orbranched chain of 1-6 carbons. Examples of aliphatic amino acids includeAla, Aib, Val, Leu, Tle, Ile, Nle, Nva, or Abu.

[0122] What is meant by “aromatic α-amino acid” is an amino acid residueof the formula

[0123] where Z₁ is a moiety containing an aromatic ring and Z₂ ishydrogen or a moiety containing an aromatic ring. Examples of sucharomatic ring-containing moieties include, but are not limited to, abenzene or pyridine ring and the following structures with or withoutone or more substituent X on the aromatic ring (where X is,independently for each occurrence, halogen, NO₂, CH₃, OCH₃, CF₃, or OH):

[0124] Other examples of an aromatic α-amino acid of the invention aresubstituted His, such as MeHis, His (τ-Me), or His (π-Me).

[0125] As used herein, “alkyl” is intended to include those alkyl groupsof the designated length in either a straight or branched configuration.Exemplary of such alkyl groups are methyl, ethyl, propyl, isopropyl,butyl, sec-butyl, tertiary butyl, pentyl, isopentyl, hexyl, isohexyl andthe like. When the term C₀-alkyl is included in a definition it isintended to denote a single covalent bond.

[0126] The term “lower alkyl” is intended to include both branched andstraight-chain alkyl groups having 1-6 carbon atoms.

[0127] As used herein, “aryl”, is intended to include any stablemonocyclic, bicyclic, or tricyclic carbon ring(s) of up to 7 members ineach ring, wherein at least one ring is aromatic. Examples of arylgroups include phenyl, naphthyl, anthracenyl, biphenyl,tetrahydronaphthyl, indanyl, phenanthrenyl, and the like.

[0128] The term “heterocyclyl”, as used herein, represents a stable 5-to 7-membered monocyclic or stable 8- to 11-membered bicyclic or stable11-15 membered tricyclic heterocyclic ring which is either saturated orunsaturated, and which consists of carbon atoms and from one to fourheteroatoms selected from the group consisting of N, O, and S, andincluding any bicyclic group in which any of the above-definedheterocyclic rings is fused to a benzene ring. The heterocyclic ring maybe attached at any heteroatom or carbon atom which results in thecreation of a stable structure. Examples of such heterocyclic elementsinclude, but are not limited to, azepinyl, benzimidazolyl,benzisoxazolyl, benzofurazanyl, benzopyranyl, benzothiopyranyl,benzofuryl, benzothiazolyl, benzothienyl, benzoxazolyl, chromanyl,cinnolinyl, dihydrobenzofuryl, dihydrobenzothienyl,dihydrobenzothiopyranyl, dihydrobenzothio-pyranyl sulfone, furyl,imidazolidinyl, imidazolinyl, imidazolyl, indolinyl, indolyl,isochromanyl, isoindolinyl, isoquinolinyl, isothiazolidinyl,isothiazolyl, isothiazolidinyl, morpholinyl, naphthyridinyl,oxadiazolyl, 2-oxoazepinyl, 2-oxopiperazinyl, 2-oxopiperidinyl,2-oxopyrrolidinyl, piperidyl, piperazinyl, pyridyl, pyridyl N-oxide,quinoxalinyl, tetrahydrofuryl, tetrahydroisoquinolinyl,tetrahydroisoquinolinyl, tetrahydro-quinolinyl, thiamorpholinyl,thiamorpholinyl sulfoxide, thiazolyl, thiazolinyl, thienofuryl,thienothienyl, thienyl, and the like.

[0129] The term “substituted” is meant to include the recited chemicalgroup (e.g., lower alkyl, aryl, cycloalkyl, etc.) substituted with oneor more of the recited substituents (e.g., halo, hydroxy, lower alkyl,etc.). The substituent may be attached to any atom in the chemicalgroup.

[0130] The abbreviation “NMe” stands for “N-methyl-”. As used herein NMeindicates that the amide nitrogen of the associated amino acid ismethylated. Thus, “NmeCpa” indicates —N(CH₃)—CH(R)—CO— where R is4-chlorophenyl, “Nme2-Nal” indicates —N(CH₃)—CH(R)—CO— where R is2-naphthyl, and so forth.

[0131] The term alkoxy is intended to include those alkoxy groups of thedesignated length in either a straight or branched configuration.Exemplary of such alkoxy groups are methoxy, ethoxy, propoxy,isopropoxy, butoxy, isobutoxy, tertiary butoxy, pentoxy, isopentoxy,hexoxy, isohexoxy and the like.

[0132] The term halogen or halo is intended to include the halogen atomsfluorine, chlorine, bromine and iodine.

[0133] When the amino acid residue is optically active, it is the L-formthat is intended unless the D-form is expressly designated.

[0134] Materials

[0135] 4-Methylbenzhydrylamine hydrochloride resin (0.25 or 0.5 mequivg⁻¹) was obtained from Advanced ChemTech Inc., Louisville, Ky. N^(α)tert-Butyloxycarbonyl (Boc) protected amino acids were purchased fromBachem Inc., Torrance, Calif., Advanced ChemTech Inc., and SynthetechInc., Albany, Oreg. The reactive side-chains of the amino acids weremasked with one of the following groups: Cys, 4-methylbenzyloxycarbonyl;Lys, 2-chlorobenzyloxycarbonyl; Thr, O-benzyl; Tyr,O-2,6-dichlorobenzyl. All reagents and solvents were ACS grade or betterand used without further purification.

[0136] Peptide Synthesis

[0137] The peptide synthesis may be summarized by the following reaction

[0138] The compounds of formula (I) can be and were synthesized on4-methylbenzhydrylamine functionalized, 1% cross-linked polystyreneresin (0.25 or 0.5 mequiv g⁻¹), in 0.25 mmol scale on an AdvancedChemTech (model 200) synthesizer, using the following protocol:deblocking, 40% TFA (2 min, 20 min); DCM wash cycle (three washes);neutralization, 10% DIEA (1 min, 5 min); DMF wash cycle; DCM wash cycle(two washes); double coupling; first with 1,3-diisopropyl carbodiimideesters (3 equiv.), 30 min in DCM; DCM wash (three washes); secondcoupling with preformed TBTU esters (3 equiv.), 90 min in DMF, with acatalytic amount of DIEA; DMF wash (one wash); DCM wash (three washes).Coupling reactions are monitored qualitatively.

[0139] N^(∝)-Protection

[0140] After deblocking the amino group at the desired methylation site,the resin was suspended in DCM (20 mL). To this suspension, collidine (3equiv.) and o-nitrobenzenesulfonyl chloride (3 equiv.) are added and themixture was shaken using Advanced ChemTech (model 200) synthesizer for 2h. Then the resin was subjected to DCM wash (2 washes) and DMF wash (3washes). Protection is monitored qualitatively by the ninhydrin test.

[0141] N^(α)-Methylation

[0142] The o-nitrobenzenesulfonamide protected resin was suspended inDMF (20 mL), to which MTBD (3 equiv.) and methyl 4-nitrobenzenesulfonateor dimethyl sulfate (for Cys¹¹) was added. The mixture was shaken usingAdvanced ChemTech (model 200) synthesizer for 0.5 h and the resin wassubjected to DMF wash (4 washes).

[0143] N^(α)-Me Deprotection

[0144] Once the desired residue was methylated, the resin was againsuspended in DMF (20 mL). DBU (3 equiv.) and 2-mercaptoethanol (3equiv.) were added to the suspension and the mixture was agitated for0.5 h in Advanced ChemTech (model 200) synthesizer. The resin was thenthoroughly washed with DMF (5 washes).

[0145] The foregoing methylation procedure worked well for all residuesexcept for D-Cys⁶, which resulted in dimethylated derivatives, (see,e.g., compounds 2 and 10.) However replacement of D-Cys⁶ with Cys⁶ gavemonomethylated peptides.

[0146] Peptide Cleavage

[0147] The peptides were cleaved from the resin support withsimultaneous side-chain deprotection by acidolysis using anhydroushydrogen fluoride containing the scavenger anisole (˜30% v/v) for 45 minat 0° C. The peptides were cyclized in 90% acetic acid (˜600 mL) with aslight excess of I₂ (15 min). Excess I₂ was then removed by the additionof ascorbic acid.

[0148] Purification

[0149] The crude peptides were purified by preparative RP-HPLC on C-18bonded silica gel using axial compression columns (Dynamax-300 Å, 5 or 8μm, 21.4×250 mm). A linear gradient elution system at a flow rate of 20mL min⁻¹ was employed: A; 0.1% TFA, B; 0.1% TFA in 80% MeCN, 20% B to50% B at 1% min⁻¹. The separations were monitored by analytical RP-HPLCat 215 nm. The fractions containing the product were pooled,concentrated in vacuo and subjected to lyophilization. Each peptide wasobtained as a fluffy white powder of constant weight by lyophilizationfrom aqueous acetic acid. The purity of the final peptides was assessedat 215 nm by analytical RP-HPLC. Analytical RP-HPLCs were recorded usinga Vydac C-18 support (4.6×250 mm, 5 μm, 300 Å pore size, LiquidSeparations Group). The linear gradient system was used at a flow rateof 1.5 mL min⁻¹: HPLC-1, A, 0.1% TFA; B, 0.1% TFA in 80% MeCN; 20% B to50% B at 1% min⁻¹; HPLC-2, C, 5% MeCN in TEAP (0.1 M, pH 3); D, 20% C inMeCN, 10% D to 70% D at 1% min⁻¹. Column eluent was monitored at 215 nm.The retention time and purity of each peptide was assessed by the RaininDynamax HPLC Method Manager. Each peptide was found to have a purityof >98%. The HPLC retention time results are given in Table 1. TABLE 1N-Methyl Analogs and Analytical Data Mass Spectrum (M − H⁺) HPLC^(c)Peptide No. N—Me Sequence Calcd.^(a) Obsd.^(b) (t_(R-1))^(d)(t_(R-2))^(e) 1 NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr- 1178.7 1179.2 18.319.3 Cys)-2-Nal-NH₂ 2 Cpa-cyclo(NmeDCys-3-Pal-DTrp-Lys-Thr- 1192.71193.4 19.6 19.3 Cys)-2-Nal-NHMe 3 Cpa-cyclo(DCys-Nme3-Pal-DTrp-Lys-Thr-1178.7 1178.9 20.3 22.5 Cys)-2-Nal-NH₂ 4Cpa-cyclo(DCys-3-Pal-NMeDTrp-Lys-Thr- 1178.7 1179.2 17.9 17.2Cys)-2-Nal-NH₂ 5 Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr- 1178.7 1178.919.2 18.7 Cys)-2-Nal-NH₂ 6 Cpa-cyclo(DCys-3-Pal-DTrp-Lys-NMeThr- 1178.71179.3 17.4 15.1 Cys)-2-Nal-NH₂ 7 Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-1178.7 1179.0 18.5 16.7 NmeCys)-2-Nal-NH₂ 8Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)- 1178.7 1179.0 20.3 19.0(Nme)-2-Nal-NH₂ 9 Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)- 1164.8 1164.717.2 17.2 Nal-NH₂ 10 Cpa-cyclo(NMeDCys-3-Pal-DTrp-Lys-Thr- 1218.9 1218.921.9 20.8 Cys)-Dip-NHMe 11 Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys- 1192.71192.3 19.9 19.7 Thr-Cys)-2-Nal-NH₂ 12Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)- 1193.8 1193.6 24.9 23.12-Nal-NH₂ 13 Tfm-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr- 1212.2 1212.2 21.420.7 Cys)-2-Nal-NH₂ 14 Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr- 1167.81168.0 16.6 14.9 Cys)-DTrp-NH₂ 15 Nal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-1183.2 1183.5 18.0 16.4 Cys)-DTrp-NH₂ 163-Pal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr- 1135.0 1134.8 11.5 9.8Cys)-DTrp-NH₂

[0150] Amino Acid Analysis

[0151] The peptides were hydrolyzed in vacuo (110° C.; 20 h) in 4 Mmethanesulfonic acid containing 0.2% 3-(2-aminoethyl)indole. (Pierce).Amino acid analyses were performed on the hydrolyzates followingderivatization with o-phthalidaldehyde reagent (Sigma Chemical Co.)using an automatic HPLC system (Rainin Instrument Co.) fitted with a100×4.6 mm, 3 μm C18 axial compression column with integral guard column(Microsorb AAAnalysis™, Type O; Rainin Instrument Co.) The derivatizedprimary amino acids were eluted using a binary gradient of buffer A;0.10 M sodium acetate containing 4.5% v/v methanol and 0.5% v/vtetrahydrofuran at pH 7.2 and buffer B; methanol. The gradient sequence;0% A at 0 min; 35% A at 16.5 min; 90% A at 30 min and 90% A at 33 min isused with a flow rate of 1.0 mL min⁻¹ at ambient temperature. Eluent ismonitored at 340 nm and integrated by the Dynamax HPLC Method Manager(Rainin). Standard retention times were as follows: Asp, 6.6 min; Arg,19.9 min; Trp, 25.4 min and Lys, 29.5 min. Each peptide of Table Iproduced the expected analytical results for the primary amino acids.Cysteine is not quantified.

[0152] Mass Spectrometry

[0153] The peptides were analyzed by matrix-assisted laserdesorption/ionization time-of-flight mass spectrometry using a LaserMat2000 mass spectrometer (Thermal Bioanalysis, San Jose, Calif.) usingα-cyano-4-hydroxycinnamic acid as the matrix with Substance P (1348.7Da) as an internal standard. In each case, the spectra consisted of amajor M−H⁺ ion peak for the internal standard, the expected analyte M−H⁺peak, and a few peaks associated with the matrix (<500 Da). The resultsare given in Table 1.

[0154] Antagonism of SRIF Inhibition of GH Release

[0155] Anterior pituitaries from adult male rats were collected anddispersed by a previously described trypsin/DNase method. (Murphy, W.A.; Taylor, J.; Moreau, J. P. and Coy, D. H., Peptide Res. 1989, 2,128-132.) The dispersed cells were diluted with sterile-filteredDulbecco's modified Eagle medium (MEM, Gibco Laboratories, Grand Island,N.Y.), which was supplemented with 2.5% fetal calf serum (Gibco), 3%horse serum (Gibco), 10% fresh rat serum (stored on ice for no longerthan 1 h) from the pituitary donors, 1% MEM nonessential amino acids(Gibco), gentamycin (10 ng mL⁻¹; Sigma) and nystatin (10,000 U mL⁻¹;Gibco). The cells were randomly plated at a density of approximately200,000 cells/well (Costar cluster 24; Rochester Scientific Co.,Rochester, N.Y.). The plated cells were maintained in the aboveDulbecco's medium in a humidified atmosphere of 95% air/5% CO₂ at 37° C.for 4-6 days. In preparation for a hormone challenge, the cells werewashed with medium 199 (Gibco, 3×1 mL). Each dose of a compound of thisinvention (6 doses/plate) was tested in triplicate wells in the presenceof 1 nM SRIF in a total volume of 1 mL medium 199 containing 1% BSA(fraction V; Sigma Chemical Co.). All wells contained GHRH(1-29)NH₂ (1nM). A GHRH(1-29)NH₂ (1 nM) stimulated control group and an SRIF (1 nM)with GHRH(1-29)NH₂ (1 nM) inhibited control group were included on eachcell culture plate. After 3 h incubation in an air/carbon dioxideatmosphere (95/5%, 3 h at 37° C.), the medium was removed and stored at−20° C. until assayed for hormone content. Growth hormone in media wasmeasured by a standard double antibody RIA using components generouslysupplied by Dr. A. F. Parlow at the National Hormone and PituitaryProgram (NHHP) Torrance, Calif.

[0156] Antagonist IC₅₀'s versus SRIF (1 nM) were calculated usingSigmaplot (Jandel Scientific, San Rafael, Calif.). Values are expressedas the mean IC₅₀ (nM)±SEM and are given in Table 2. TABLE 2 BindingAffinities (K₁) for Cloned Human sst₁₋₅ Receptors and Antagonist DataAntagonist N^(α)-Methylation K₁ ^(a) ± SEM (nM) IC₅₀ ± SEM Peptide No.site hsst₁ hsst₂ hsst₃ hsst₄ hsst₅ (nM)^(b) SRIF-14 N/A^(c)  2.0 ± 0.350.25 ± 0.03 1.2 ± 0.2 2.0 ± 0.3 1.4 ± 0.3 N/A^(c) SRIF-28 N/A^(c) 1.9 ±0.4 0.31 ± 0.06 1.3 ± 0.3 5.4 ± 2.5 0.4 ± 0.1 N/A^(c)  1 Cpa⁵ 1000  36 ±7.6 330 ± 126 1000 40.1 ± 18.8 7.8 ± 2.7  2^(d) D-Cys⁶ 1000 89.0 ± 8.0 576 ± 47  1000 106 ± 36  nd^(e)  3 3-Pal⁷ 1000 189.0 ± 35   450 ± 1321000 1000 nd^(e)  4 D-Trp⁸ 1000 51.8 ± 2.6  390 ± 114 1000   93 ± 17.7nd^(e)  5 Lys⁹ 1000 17.1 ± 5.5  66.0 ± 5.8  1000 5.98 ± 0.91 0.73 ± 0.33 6 Thr¹⁰ 395 ± 202 1000  315 ± 12.5  1000 88.5 ± 45.7 nd^(e)  7 Cys¹¹1000 810 68.7 ± 4.7   575 161 ± 52  nd^(e)  8 2-Nal¹² 1000 197 ± 55 1000 1000 1000 nd^(e)  9 N/A^(c) 1395 12.1 ± 1.9  38.2 ± 2.4  1000  140± 4.6  2.6 ± 0.7 10^(d) D-Cys⁶ 1000  117 ± 24.6 584 ± 305 1000 766 ± 110nd^(e) 11 DTrp⁸ 1000 9.33 ± 0.62  140 ± 10.4 1000 112 ± 19   2.5 ± 0.20& Lys⁹ 12 Lys⁹ 1000 5.51 ± 1.85 115.1 ± 16.9  1000 70.7 ± 25.8 0.53 ±0.17 13 Lys⁹ 1000 11.3 40.2 1246 45.5 nd^(e) 14 Lys⁹ 1000 5.45 ± 0.3 91.4 ± 11.9 1000  101 ± 14.1 11.6 ± 4.2  15 Lys⁹ 1000 27.3 ± 1.45  148 ±13.2 1000  176 ± 65.1   96 ± 13.8 16 Lys⁹ 1000 24.7 ± 1.61  537 ± 44.81000  313 ± 4.1  287 ± 138

[0157] Functional Expression of the Cloned Human Somatostatin Receptors

[0158] The genomic clones containing the human somatostatin receptors(hsstr₁₋₅) (Yamada, Y., et al. al., Proc. Natl. Acad. Sci. USA. 1992,89, 251-255; Yasuda, K., et al., J. Biol. Chem. 1992, 267, 20422-20428;Yamada, Y., et al., Mol. Pharmacol. 1992, 42, 2136-2142; Rohrer, L., etal., Proc. Natl. Acad. Sci. USA. 1993, 90, 4196-4200.), were kindlyprovided by Dr. Graeme I. Bell (University of Chicago). The hsstr₁,hsstr₂, hsstr₃, hsstr₄ and hsstr₅ cDNAs were isolated as a 1.5-kbPstI-XmnI fragment, 1.7-kb BamHI-HindIII fragment, 2.0-kb NcoI-HindIIIfragment, 1.4-kb NheI-NdeI fragment, and a 1.2-kb HindIII-XbaI fragment,respectively, each containing the entire coding region of thefull-length receptors. These fragments were independently subcloned intothe corresponding restriction endonuclease sites in the mammalianexpression vector pCMV5, downstream from the human cytomegalovirus (CMV)promoter, to produce the expression plasmids pCMV5/hsstr₁, pCMV5/hsstr₂,pCMV5/hsstr₃, pCMV5/hsstr₄ and pCMV5/hsstr₅. For transfection intoCHO-K1 cells, a plasmid, pRSV-neo (American Type Culture Collection,Rockville, Md.), carrying the neomycin mammalian cell selectable markerwas added.

[0159] Receptor Expression and Transfection

[0160] Transfections were performed by the calcium phosphate method.CHO-K1 cells are maintained in α-minimum essential medium (α-MEM; Gibco)supplemented with 10% fetal calf serum and transfected with each of theexpression plasmids using calcium phosphate precipitation. Clones thathad inherited the expression plasmid were selected in α-MEM supplementedwith 500 μg mL⁻¹ of geneticin (G418; Gibco). Independent CHO-K1 cloneswere picked by glass-ring cloning and expanded in culture in theselective media. Membranes were prepared from the isolated clones andhsstr expression was initially assessed for binding with[¹²⁵I]Tyr¹¹-SRIF and [¹²⁵I]MK-678 (for sstr₂).

[0161] Radioligand Binding Assays

[0162] Cell membranes of the 5 receptor types were obtained fromhomogenates (Polytron setting 6, 15 sec) of the corresponding CHO-K1cells, in ice-cold Tris-HCl (50 mM) and centrifuged (39000 g, 10 min×2),with an intermediate resuspension in fresh buffer. The final pellets areresuspended in Tris-HCl (10 mM) for assay. Aliquots of the membranes areincubated (30 min at 37° C.) with 0.05 nM [¹²⁵I]Tyr¹¹-SRIF (types1,3,4,5) or [¹²⁵I]MK-678 (type 2) in 50 mM HEPES (pH 7.4) containing BSA(10 mg mL⁻¹); MgCl₂ (5 mM), Trasylol (200 kIU mL⁻¹), bacitracin (0.02 mgmL⁻¹), and phenylmethanesulfonyl fluoride (0.02 mg 1 mL⁻¹). The finalassay volume is 0.3 mL and incubations are terminated by rapidfiltration through GF/C filters pre-soaked in 0.3% poly(ethylenimine)using a Brandel rapid filtration module. Each tube and filter is thenwashed with aliquots of cold buffer (3×5 mL).

[0163] Specific binding is. defined as the total radioligand bound minusthat bound in the presence of 1.0 μM SRIF. The following totalradioligand binding and non-specific binding (nsb) values are typicallyobtained with these assay systems: hsstr₁, 7000 cpm total versus 3500cpm nsb; hsstr₂, 9000 cpm total versus 1000 cpm nsb; hsstr₃, 8000 cpmtotal versus 1000 cpm nsb; hsstr₄, 6000 cpm total versus 3500 cpm nsb;and hsstr₅, 7500 cpm total versus 3500 cpm nsb. The binding affinitiesare expressed as K_(i) values±SEM (nM) for each of the five receptorsubtypes and are given in Table 2.

[0164] Type 5 Mediated Intracellular Ca²⁺ Mobilization

[0165] CHO-K1 cells, expressing the human sst5 receptor, were harvestedby incubating in a 0.3% EDTA/phosphate buffered saline solution (25°C.), and washed twice by centrifugation. The washed cells wereresuspended in Hank's -buffered saline solution (HBSS) for loading ofthe fluorescent Ca²⁺ indicator Fura-2AM. Cell suspensions ofapproximately 10⁶ cells/ml were incubated with 2 μM Fura-2AM for 30 minat about 25° C. Unloaded Fura-2AM was removed by centrifugation twice inHBBS, and the final suspensions were transferred to a spectrofluorometer(Hitachi F-2000) equipped with a magnetic stirring mechanism and atemperature-regulated cuvette holder. After equilibration to 37° C., thesomatostatin peptides were added for measurement of intracellular Ca²⁺mobilization. The excitation and emission wavelengths were 340 and 510nm, respectively.

[0166] Exemplary data appears in FIG. 1 which depicts results from theimmediately foregoing assay using Analog 5 as the Test Compound.

[0167] Molecular Modeling

[0168] Molecular modeling was performed on a Silicon Graphics Indigo²High Impact 10000 computer, using SYBYL molecular modeling software,version 6.6, (Tripos Associates Inc., St. Louis Mo., USA), with theKollman all atom force field. (Weiner, S. J., et al., J. Comp. Chem.1986, 7, 230-252.) The PDB files for the three solution NMR structuresof the initial compound Sandostatin/Octreotide;D-Phe⁵-c[Cys⁶-Phe⁷-D-Trp⁸-Lys⁹-Thr¹⁰-Cys¹¹]-Thr¹²-ol (1 SOC and 2SOC)were obtained from the PDB database. These structures were imported intoSYBYL 6.6 and mutated to form the N-methylated compounds based on analog9. The Kollman partial atomic charges were loaded from the monomerdictionary. The structures were optimized by annealing the mutatedresidue and then by full energy minimization using the conjugategradient algorithm to a final root mean square (rms) gradient of ≦0.01Kcal molÅ⁻¹. A distance-dependent dielectric function (McCammon, J. A.,et al., Biochem. 1979, 18, 927-942) was employed together with thedefault settings for all the other minimization options. The results aredetailed in Table 3. TABLE 3 Kollman all atom energy change onsequential methylation of each residue of Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂ (analogue 9) in each of the threesolution conformations expressed as Kcal mol⁻¹. Methylation Analogue NoSite I II III 1 NMeCpa⁵ −2 0.4 −1 2 NMeDCys⁶, NHMe 17 12 31 3 NMe3-Pal⁷7 17 6 4 NMeDTrp⁸ 5 6 5 5 NMeLys⁹ 6 6 5 6 NMeThr¹⁰ 16 10 12 7 NMeCys¹¹12 14 23 8 Nme-2-Nal¹² 4 19 7

[0169] Other Embodiments

[0170] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, that theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the claims.

What is claimed is:
 1. A compound of the formula (I),A¹-cyclo{D-Cys-A²-D-Trp-A³-A⁴-Cys}-A⁵-Y¹,  (I) wherein: A¹ is anoptionally substituted aromatic α-amino acid; A² is an optionallysubstituted aromatic α-amino acid; A³ is Dab, Dap, Lys or Orn; A⁴ isβ-Hydroxyvaline, Ser, Hser, or Thr; A⁵ is an optionally substituted D-or L-aromatic α-amino acid; and Y¹ is OH, NH₂ or NHR¹, where R¹ is(C₁₋₆)alkyl; wherein each said optionally substituted aromatic α-aminoacid is optionally substituted with one or more substituents eachindependently selected from the group consisting of halogen, NO₂, OH,CN, (C₁₋₆)alkyl, (C₂₋₆)alkenyl, (C₂₋₆)alkynyl, (C₁₋₆)alkoxy, Bzl, O-Bzl,and NR⁹R¹⁰, where R⁹ and R¹⁰ each is independently H, O, or (C₁₋₆)alkyl; and wherein the amine nitrogen of each of amide peptide bond andthe amino group of A¹ of formula (I) is optionally substituted with amethyl group, provided that there is at least one said methyl group; ora pharmaceutically acceptable salt thereof.
 2. A compound according toclaim 1, wherein: A¹ is Cpa, 1-Nal, 2-Nal, 2-Pal, 3-Pal, 4-Pal, Phe,Tfm, Tyr or Tyr(I); A² is 2-Pal, 3-Pal, 4-Pal, Phe, Tyr or Tyr(I); andA⁵ is Dip, 1-Nal, 2-Nal, 2-Pal, 3-Pal, 4-Pal, Phe or D-Trp; or apharmaceutically acceptable salt thereof.
 3. A compound according toclaim 2, wherein A¹ is Cpa.
 4. A compound according to claim 2, whereinA³ is NMeLys.
 5. A compound according to claim 1, wherein said compoundis according to the formula:NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(NMeDCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NHMe;Cpa-cyclo(DCys-NMe3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-NMeDTrp-Lys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-Lys-NMeThr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-NMeCys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-Nme2-Nal-NH₂;Cpa-cyclo(NMeDCys-3-Pal-DTrp-Lys-Thr-Cys)-Dip-NHMe;Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Tfm-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;Nal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or3-Pal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or apharmaceutically acceptable salt thereof.
 6. A compound according toclaim 4, wherein said compound is according to the formula:NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂;Nal-cyclo(DCys-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or3-Pal-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or apharmaceutically acceptable salt thereof.
 7. A compound according toclaim 5, wherein said compound is according to the formula:NmeCpa-cyclo(DCys-3-Pal-DTrp-Lys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-3-Pal-NMeDTrp-NMeLys-Thr-Cys)-2-Nal-NH₂;Cpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂; orCpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-DTrp-NH₂; or apharmaceutically acceptable salt thereof.
 8. A compound according toclaim 6, wherein said compound is according to the formula:Cpa-cyclo(DCys-3-Pal-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂; orCpa-cyclo(DCys-Tyr-DTrp-NMeLys-Thr-Cys)-2-Nal-NH₂; or a pharmaceuticallyacceptable salt thereof.
 9. A method of binding one or more somatostatinsubtype receptors −1, −2, −3, −4 and −5, which comprises the step ofcontacting a compound of claim 1 or a pharmaceutically acceptable saltthereof with one or more of said somatostatin subtype receptors.
 10. Amethod of binding one or more somatostatin subtype receptors −1, −2, −3,−4 and −5 in a human subject or other animal subject, which comprisesthe step of administering an effective amount of a compound of claim 1or a pharmaceutically acceptable salt thereof to a subject in needthereof.
 11. A method of eliciting an antagonist effect from a cell,wherein said cell comprises one or more somatostatin receptors, saidmethod comprising contacting said cell with an effective amount of acompound of claim 1 or a pharmaceutically acceptable salt thereof.
 12. Amethod of eliciting a somatostatin antagonist effect in a human oranimal subject, which comprises the step of administering an effectiveamount of a compound of claim 1 or a pharmaceutically acceptable saltthereof to a subject in need thereof.
 13. A method of promoting therelease of growth hormone in a human or animal subject, which comprisesadministering to said subject an effective amount of a compoundaccording to claim 1 or a pharmaceutically acceptable salt thereof. 14.A method of promoting the release of insulin in a human or animalsubject in need thereof, which comprises administering to said subjectan effective amount of a compound according to claim 1 or apharmaceutically acceptable salt thereof.
 15. A method of enhancingwound healing in a human or animal subject in need thereof, whichcomprises administering to said subject an effective amount of acompound according to claim 1 or a pharmaceutically acceptable saltthereof.
 16. A method of promoting angiogenesis in a human or animalsubject in need thereof, which comprises administering to said subjectan effective amount of a compound according to claim 1 or apharmaceutically acceptable salt thereof.
 17. A method of imaging cellshaving somatostatin receptors which comprises contacting said cells withan effective amount of a compound according to claim 1, or apharmaceutically acceptable salt thereof, which comprises Tyr(I).
 18. Amethod of treating a disease or condition in a human or other animalsubject in need thereof, which comprises the step of administering aneffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof to said subject, wherein said disease orcondition is selected from the group consisting of short stature,cachexia, wasting, type 2 diabetes, and poor circulation